CN109265553A

CN109265553A - A kind of fusion protein of cytoglobin and Sipunculus nudus plasmin

Info

Publication number: CN109265553A
Application number: CN201810968815.7A
Authority: CN
Inventors: 许瑞安; 李招发; 陈磊; 肖玲慧; 马国兴; 崔秀灵
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-08-23
Filing date: 2018-08-23
Publication date: 2019-01-25
Anticipated expiration: 2038-08-23
Also published as: CN109265553B

Abstract

The invention discloses the fusion proteins of a kind of cytoglobin and Sipunculus nudus plasmin, the present invention further provides the preparation methods of fusion protein and related strain to construct, the present invention is expanded by strain culturing, separate supernatant, sulfuric acid precipitation, Sephadex G-25 desalination, DEAE stepwise elution purifies to have obtained the fusion protein, the optimal pH range of fusion protein of the invention is in 40 DEG C or less pH 5.0~9.0, enzyme activity keeps relative stability, and PMSF and Cu2+ are larger to the activity influence of the fusion protease.The fusion protein has the characteristic of targeting fibrin degradation original, and degradation sequencing is α chain > β chain > γ chain.Fusion protein of the invention has better thrombolytic effect compared with urokinase in thrombus experiment.

Description

A kind of fusion protein of cytoglobin and Sipunculus nudus plasmin

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of cytoglobin merges egg with Sipunculus nudus plasmin It is white, their preparation method and its effect.

Background technique

Thrombotic disease is a kind of common cardiovascular and cerebrovascular disease, and disease incidence is in first of various diseases.The whole world About 2,000,000,000 dollars of potential market of thrombolytics needed for annual.And marine active substance is because of its structure novel, pharmacological activity height etc. The hot spot that advantage is increasingly becoming Recent study identifies to come from ocean there are also more and more fibrinolytic substances, wherein very Mostly can not only plasminogen activation can also direct solution fibrin.In clinical application process, if adding fibrinolysin outside While activator plasminogen activation, the enzyme of external source, energy directly fibrin degradation is used, so that it may extraly reinforce Plasma fibrinolytic activity, so that it may to reach more preferably thrombolytic effect.Although currently can be applied to clinical Studying On Marine Cardiovascular Drugs Also seldom, the research work of the gene cloning and expression about marine organisms source fibrinolysin is also rarely reported, but this is worth The field having a extensive future must be opened up.

Summary of the invention

Cytoglobin (Cytoglobin, Cygb) is a kind of newly discovered hexa-coordinate ball egg containing prosthetic heme group It is white.

Ocean Sipunculus nudus plasmin (referred to as: PL) is homogenized from ocean Sipunculus nudus glandula digestive body fluid, and grease removal is filtered, Freeze-drying, saltouts, desalination, and the albumen that DEAE chromatography and gel permeation chromatography obtain, N-terminal portion amino acid sequence is Seq.2, point Son amount 15KDa, Sipunculus nudus plasmin have good effect in terms of antithrombotic and apoplexy prevention and treatment.

The purpose of the present invention is to provide the fusion proteins of a kind of high efficient expression cytoglobin and Sipunculus nudus plasmin And the genetic engineering bacteria strain of Sipunculus nudus plasmin.

It is a further object of the present invention to provide the fusion proteins of born of the same parents' globin and Sipunculus nudus plasmin, and are melted by this Hop protein, the method that cracking prepares Sipunculus nudus plasmin, and verify the fusion protein of born of the same parents' globin and Sipunculus nudus plasmin With the curative effect of medication of Sipunculus nudus plasmin.

The specific technical solution of the present invention is as follows:

A kind of fusion protein of cytoglobin and Sipunculus nudus plasmin, entitled CYGB-PL, the fusion protein have Amino acid sequence shown in SEQ ID NO:1.

Fusion protein of the present invention, which is characterized in that Sipunculus nudus plasmin has ammonia shown in SEQ ID NO:2 Base acid sequence.

Fusion protein of the present invention, which is characterized in that cytoglobin has amino acid shown in SEQ ID NO:3 Sequence.

Fusion protein of the present invention, which is characterized in that the fusion protein contains flexible polypeptide, and flexible polypeptide has Amino acid sequence shown in SEQ ID NO:4.

Fusion protein of the present invention, which is characterized in that the fusion protein contains fibrin ferment pentapeptide, fibrin ferment pentapeptide tool There is amino acid sequence shown in SEQ ID NO:5.

A kind of engineering bacteria, entitled pPIC-9-CYGB-PL-GS115 are expressed as fusion protein of the present invention CYGB-PL。

A kind of recombinant vector, entitled pPIC-9-CYGB-PL, wherein pPIC9 carrier contains AOX1 promoter, need to be through first It is alcohol-induced.The recombinant vector, preparation process include through BglII single endonuclease digestion, and electricity is transferred to yeast GS115.

The preparation method of fusion protein of the present invention, the culture including engineering bacteria, the separation of fusion protein, purifying step Suddenly, especially by sulfuric acid precipitation, Sephadex G-25 desalination, DEAE stepwise elution purifies to obtain.

The invention also includes the pharmaceutical compositions containing fusion protein.And fusion protein of the present invention is preparing antithrombotic Application in drug.

The present invention further provides the coding bases of the Codocyte globin and the fusion protein of Sipunculus nudus plasmin Cause has nucleotide sequence shown in SEQ ID NO:6.

The encoding gene for wherein encoding Sipunculus nudus plasmin, has nucleotide sequence shown in SEQ ID NO:7.

The wherein encoding gene of Codocyte globin has nucleotide sequence shown in SEQ ID NO:8.

The encoding gene for wherein encoding flexible polypeptide, has nucleotide sequence shown in SEQ ID NO:9.

The encoding gene for wherein encoding fibrin ferment pentapeptide, has nucleotide sequence shown in SEQ ID NO:10.

In a preferred embodiment of the invention, include the following steps: construction recombination plasmid pPIC9-CYGB-PL, And carry out bacterium colony PCR and double digestion verifying.As shown in Figure 1, 2, 3.

It in a preferred embodiment of the invention, further include following steps: building pPIC9-CYGB-PL-GS115 ferment Mother strains, as shown in Figure 4.

In a preferred embodiment of the invention, CYGB-PL protein SDS-PAGE detecting step is as follows: when will be different Between the fermented supernatant fluid of section carry out SDS-PAGE electrophoresis, and the expression quantity of CYGB-PL albumen is analyzed, as shown in Figure 5.

It in a preferred embodiment of the invention, is pPIC9-CYGB-PL-GS115 yeast strain amplification cultivation, with Fermented supernatant fluid is extracted afterwards and carries out ammonium sulfate precipitation, G-25 desalination, DEAE gradient elution, and purifying obtains fusion protein, goes forward side by side The verifying of row activity and purity analysis.As shown in Fig. 6,7,8,9.

In a preferred embodiment of the invention, to carry out physicochemical properties detection to fusion protein of the present invention, Include: to be placed in the enzyme under different pH and different temperatures, measures its optimal pH and optimal reactive temperature.Different inhibition is added Agent and metal ion measure the influence of different inhibitor and metal ion to the enzymatic activity, as shown in Figure 10,11,12.

In a preferred embodiment of the invention, the enzyme is measured using heating flat band method to fusion protein of the present invention Direct Fibrinolytic Activity, and urokinase is added as negative control.As shown in figure 13.

In a preferred embodiment of the invention, fusion protein of the present invention is incubated for fibrinogen, is taken Different time sections sample carries out SDS-PAGE electrophoresis.As shown in figure 14.

In a preferred embodiment of the invention, fusion protein of the present invention is used for using Mouse whole blood grumeleuse external Urokinase is added as positive control in thrombolytic experiment, and physiological saline calculates the dissolution rate of clot and observe not as negative control With the burst size and cellular morphology of the cell in period solution.As shown in Figure 15,16,17.

In a preferred embodiment of the invention, fusion protein solution of the present invention is injected intraperitoneally small into thrombus model In mouse, and urokinase is added as positive control, as negative control, the length for counting mouse tail thrombus becomes physiological saline Change.As shown in Figure 19,20.

The beneficial effects of the present invention are: gained genetic engineering bacteria strain fusion protein expression with higher, gained melt Hop protein has compared with the higher biological activity of Sipunculus nudus plasmin, has good thrombolytic effect, and have targeting molten Solve the effect of fibrinogen.

Detailed description of the invention

Fig. 1 is that (M:2000DNA Marker 1: the single colonie 2 of random picking: random picking obtains list to bacterium colony PCR qualification figure Bacterium colony)

Fig. 2 is that XholI and EcoRI double digestion plasmid pPIC9-CYGB-PL schemes (M:2000DNA Marker 1: plasmid PPIC9-CYGB-PL 2: plasmid pPIC9-CYGB-PL)

Fig. 3 is the part sequencing result figure of plasmid pPIC9-CYGB-PL

Fig. 4 is that PCR identifies that pPIC9-CYGB-PL is transferred to GS115 and schemes (M:2000DNA Marker 1: positive control (plasmid PET22b-CYGB-PL) 2-19: the single colonie selected at random)

Fig. 5 SDS-PAGE detection CYGB-PL albumen (M: albumen Marker 1: induction preceding 2: induction 12h 3:24h 4: 36h 5:48h 6:60h 7:72h 8:83h)

Fig. 6 is ammonium sulfate precipitation curve graph

Fig. 7 is the supernatant of ammonium sulfate precipitation and the fibrinolytic figure of precipitating

Fig. 8 is DEAE Sepharose Fast Flow anion-exchange chromatography figure

Fig. 9 is CYGB-PL PAGE gel electrophorogram.

Figure 10 is influence of the pH value to CYGB-PL stability.

Figure 11 is influence of the temperature to enzyme activity in 1h.

Figure 12 is the THERMAL STABILITY of enzyme.

Figure 13 is the influence of different metal ions and enzyme inhibitor to enzyme activity.

Figure 14 is fiber plate Activity determination CYGB-PL activity figure (1: not inducing；2: induction 12h；3: induction is for 24 hours；4: luring Lead 36h；5: induction 48h；6: induction 60h；7: induction 72h；8: induction 77h；9:1000U/mL UK).

Figure 15 is that CYGB-PL is mode of action figure to fibrinogen.

Figure 16 is external thrombolytic experiment (thrombolysis after clot weight in wet base 100mg, 0h, 1h, 2h, 3h).

Figure 17 is external thrombolytic experiment red blood cell microscopic examination result figure (200X).

Figure 18 Mouse whole blood clot dissolution rate figure.

Mouse tail thrombus length after 72h is administered in Figure 19.

Figure 20 different dosing group mouse tail thrombus length statistical chart.

Specific embodiment

Technical solution of the present invention is further explained and described below by way of specific embodiment.

The building of 1 recombinant plasmid pPIC9-CYGB-PL of embodiment

(1) plasmid pET22b-CYGB-PL is that template PCR amplifications go out CYGB-PL sequence, and system is as follows:

Reaction condition: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 45s, 53 DEG C of annealing 30s, 72 DEG C of extension 1min10sec are followed Ring 5 times；It last 72 DEG C, 10min, is saved in 4 DEG C.50 μ L of PCR product is taken to be mixed evenly with 10 μ 6 × loading of L buffer； 90V electrophoresis 25min, electrophoresis, which terminates to take out to observe under gel to gel image analyser, takes pictures, and glue recycles target fragment.Glue returns It receives；

(2) double digestion of pPIC9 plasmid and glue recycling CYGB-PL DNA fragmentation

Double digestion system is as follows:

Reaction condition: 37 DEG C of water-baths recycle digestion products, -20 DEG C of short time overnight (12h), to end of reaction gel electrophoresis It saves.Glue recycling；

(3) plasmid pPIC9 is reacted with the connection of target gene CYGB-PL

Linked system is as follows:

Reaction condition: 16 DEG C connect overnight (12h), are transferred to by heat shock method thin to E.coli DH5 α competence Born of the same parents enable pPIC9-CYGB-PL recombinant plasmid largely to be expanded.

Embodiment 2 recombinates the bacterium colony PCR verifying of pPIC9-CYGB-PL

Identification system is as follows:

Reaction condition: 94 DEG C of initial denaturations, 4min recycle 94 DEG C of denaturation 45s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min10s, Circulation 25 times；Last 72 DEG C, 10min, 4 DEG C preservations.5 μ L of PCR product and 1 μ L 6 × loading buffer buffer is taken to mix Loading, 90V electrophoresis 25min, electrophoresis, which terminates to take out to observe under gel to gel image analyser, takes pictures.

The double digestion and PCR of 3 recombinant plasmid pPIC9-CYGB-PL of embodiment is verified

It is that positive single colonie is inoculated in LB culture medium of the 5mL containing Amp by qualification result, 37 DEG C, the training of 250rpm shaking table Support 13h or so rear extraction plasmid.Plasmid double digestion system is as follows:

Reaction condition: then 37 DEG C of water-bath 7h carry out gel electrophoresis and photograph to record result.

Plasmid PCR system is as follows:

By plasmid double digestion and plasmid PCR identification be positive findings single colonie send to Nanjing Jin Sirui Co., Ltd into Row sequencing.

The building of 4 pichia pastoris engineered strain GS115-CYGB-PL of embodiment

The preparation of competent yeast:

(1) it by Pichia pastoris GS115 plate streaking and YPD solid medium, cultivates 2 days or so.

(2) the good GS115 single colonie of picking epidemic situation comparison is inoculated in 5mL YPD fluid nutrient medium, in 28 DEG C, 250rpm Shaken cultivation 18 hours or so.

(3) 500 μ L bacterium solutions is taken to be seeded in 50mL YPD fluid nutrient medium, in 28 DEG C, the vibration of 250rpm shaking table is cultivated, Until OD600=1.0~1.3.

(4) bacterium solution is transferred to 50ml centrifuge tube, and 1500g, 4 DEG C of centrifugation 5min abandon supernatant.

(5) thallus is resuspended in 50mL ice sterile water, and 1500g, 4 DEG C of centrifugation 5min abandon supernatant.Bacterium is resuspended in 25ml ice sterile water Body, 1500g, 4 DEG C of centrifugation 5min abandon supernatant.

(6) suspension thalline, 1500g, 4 DEG C of centrifugation 5min abandon supernatant to 5ml ice 1M sorbierite again.

Again suspension thalline, every pipe dispense 80-100 μ l, are stored in -80 DEG C of refrigerators, about 2 (7) 500 μ l 1M sorbierites It can be used in week.

The linearisation of recombinant plasmid pPIC9-CYGB-PL

Reaction system is as follows:

Reaction condition: overnight (12h), nucleic acid electrophoresis recycles biggish gel fragment for 37 DEG C of water-baths.

Linearization plasmid electricity is rotated into GS115 cell

It is electroporated that steps are as follows:

(1) it is gone to after taking 10 μ L linearization plasmids and GS115 Pichia pastoris competent cell to mix and takes ice pre-cooling in advance In the electroporated cup of 0.2cm.

(2) by electrotransformation cup ice bath 15min or so.

(3) it is voltage 1.5kV that electricity, which turns condition,；25 μ F of capacitor；200 Ω of resistance.The electric shock time is 4~10msec.

(4) after shocking by electricity, the 1M sorbitol solution of 1mL ice pre-cooling is added into cup immediately, is gone to after mixing In the sterile centrifugation tube of 1.5mL.

(5) 28 DEG C, shaken cultivation about 1h under the conditions of 150rpm.

(6) thallus suspension is coated on MD plate, every 200 μ L or so is coated with one flat plate.

(7) after bacterium solution is completely absorbed, plate inversion is incubated in 28 DEG C of incubators and is cultivated 2-3 days, until single Bacterium colony occurs.

Extract pastoris genomic dna identification positive transformant (GS115-pPIC9-CYGB-PL)

The extraction step of pastoris genomic dna is as follows:

1, yeast cells is taken (to be no more than 5 × 10⁷Ce l1s), 12000rpm is centrifuged 1min, as far as possible absorption supernatant.

2, yeast cell wall is abolished: 470ul sorbierite Buffer being added into yeast thallus.Abundant suspension thalline, adds Enter 25ul yeast wall breaking enzyme and 5ul beta -mercaptoethanol, mixes well.During which 30 DEG C of processing 1-2h can overturn centrifuge tube and mix number It is secondary.

3,12000rpm is centrifuged lmin, abandons supernatant, collects precipitating.

4,200ul solution A is added into precipitating, sufficiently suspend precipitating, and the RNase A of 20ul is added into suspension (10mg/ml), is sufficiently mixed by inversion, and is placed at room temperature for 10min.

5, the Proteinase K (10mg/ml) of 20ul is added, is sufficiently mixed by inversion.65 DEG C of water-baths digest 15-30min, period of digestion Between can overturn centrifuge tube mix for several times, until treatments of the sample is complete.

6,200ul solution B is added, adds 200ul dehydrated alcohol, is sufficiently mixed by inversion, it is at this time it is possible that cotton-shaped Precipitating, does not influence the extraction of DNA, solution and flocculent deposit can all be added in adsorption column, be placed at room temperature for 2min.

7,12000rpm is centrifuged 2min, abandons waste liquid, adsorption column is put into collecting pipe.

8,600ul rinsing liquid (please first checking whether that dehydrated alcohol is added in oneself before use) is added into adsorption column. 12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collecting pipe.

9,600ul rinsing liquid is added into adsorption column, 12000rpm is centrifuged lmin, abandons waste liquid, adsorption column is put into collection Guan Zhong.

10,12000rpm is centrifuged 2min, and adsorption column opening is placed in room temperature or 50 DEG C of incubators are placed several minutes, it is therefore an objective to will Remaining rinsing liquid removal in adsorption column, otherwise the ethyl alcohol in rinsing liquid will affect subsequent experimental such as digestion, PCR etc..

11, adsorption column is put into a clean centrifuge tube, to the hanging 50-200ul that is added dropwise in adsorbed film center through 65 DEG C The eluent of water-bath preheating, is placed at room temperature for 5min, and 12000rpm is centrifuged lmin.

12, centrifugation gained eluent adds in adsorption column, and 12000rpm is centrifuged 2min, and the gene of high quality can be obtained Group DNA.

Take the genomic DNA of extraction for template PCR identification, system is as follows:

Reaction condition: 94 DEG C of initial denaturations, 4min；94 DEG C of denaturation 45s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 25 are followed Ring；It last 72 DEG C, 10min, is saved in 4 DEG C.5 μ l PCR products and 1 μ 6 × loading of l buffer are taken to mix loading, 90V Electrophoresis 25min, taking-up gel, which is placed under gel image analyser to take pictures, observes result.

The store method of strain

It is that positive strain carries out conservation by qualification result, picking single colonie is connected in 5mL YPD fluid nutrient medium, and 28 DEG C, it cultivates under the conditions of 250rpm to OD₆₀₀Value be 2~6, every 800 μ l bacterium solution add the glycerol of 200 μ l 80% to 1.5mL sterilize from It is stored in heart pipe, after sealing in -80 DEG C of refrigerators.

5 fermentor technology of embodiment prepares fusion protein

The preparation of fermentation seed liquid

Oese picking contains the Yeast engineering bacteria of pPIC9-CYGB-PL plasmid, and plate streaking is inverted in 28 in MD plate DEG C constant incubator in culture.After constant temperature incubation 48 hours, then from the good positive Dan Ke of picking growth conditions on MD plate Grand bacterium colony is inoculated in the test tube of the culture medium containing 5mlYPD, and 28 DEG C, 250r/min is cultivated 14-18 hours, until A₆₀₀When for 2-6, It is seeded in 200mlBMG culture medium by volume fraction 1%.28 DEG C, 250r/min shaking table culture 16-24h, until A₆₀₀When for 2-6, And just to can be used as fermentation tank seed liquor without microbiological contamination spare for microscopy.

The preparation of fermentor

It prepares 2L basal salt media (BSM) to pour into fermentation tank, entire tank body goes out after outer siphunculus road is mounted and sealed Bacterium.It after sterilizing, is cooled to room temperature to tank body, it is connect with the recirculated water of temperature-adjustable to adjust temperature, meanwhile, even Connect device of air, condenser pipe, stirring rotator etc..Then pH electrode is corrected, installation then will to designated position after eluting alcohol Speed of agitator rises to 500r/min, after stirring 30min, corrects dissolved oxygen electrode and installs.Thereafter, by the way that ammonium hydroxide is added dropwise by culture medium PH is adjusted to 5.0, and adjusts ventilatory capacity 5L/min, the defoaming agent that sterilized is added dropwise, flame can be passed through by being down to 28 DEG C to pot temperature The method of inoculation pours into fermentation seed liquid in fermentor, and 15mL biotin and 15mL microelement is added.

Methanol induction protein expression

Yeast is exponentially grown at the beginning in the fermenter, and oxygen dissolving value (DO, Dissolved oxygen) is caused to start not The decline stopped.Incipient stage is in order to maintain DO value 20% to 40% or so, because being conducive to yeast with this condition with appropriate Speed grown, it is therefore desirable to situation of change is obtained according to oxygen dissolving value and constantly regulate revolving speed.When yeast growth to left and right for 24 hours When, DO value can fly up and be maintained at 100% or so, this is because the glycerol in culture medium has been consumed completely, yeast There is no utilizable carbon source, respiratory metabolism slows down, and oxygen dissolving value is caused to rise.It should keep logical oxygen and revolving speed constant at this time, make ferment Glycerol in culture medium was utilized in starvation 2 hours or so and is finished by mother, the interference for avoiding glycerol from expressing methanol induction. Then start to add methanol, as the addition DO value of methanol can start slowly to decline, and methanol flow rate is higher, the meeting of DO decline It is increasing.In the case where methanol flow rate is constant, DO value will rest on one and be maintained in a more stable range It protects.It needs to carry out sensitivity Detection during methanol induction, exactly by stopping adding methanol, if in the meeting quickly of DO value It rises and then illustrates that methanol does not add excess, illustrate that methanol supply is excessive if DO value does not change the apparent variation of generation, at this time It needs to stop immediately the supply of methanol or reduces the flow velocity of methanol.During this time, it is also noted that methanol cannot add excess.It cuts For note during entire methanol induction, methanol, which cannot be added, excessively prevents yeast to be poisoned.The pH value of fermentor is set in 6.5 left sides The right side is adjusted by adding ammonium hydroxide automatically.In entire fermentation process, every mistake will add the microelement of 15mL for 24 hours With the biotin of 15mL, yeast is maintained to be in relatively better growth conditions.Entire fermentation process probably at 96 hours or so, It needs that every mistake during a sample and methanol induction is taken to need within 12 hours to take a sample, the life to yeast before not induced Long status carries out and the expression of albumen is analyzed.

The detection of index of correlation in 6 thallus Induction Process of embodiment

(1) thallus weight in wet base and A₆₀₀

The measurement of thallus weight in wet base: weighing the weight of centrifuge tube before sampling, takes 1ml bacterium solution in centrifuge tube later, and 4 DEG C, 5000r/min is centrifuged 5min.Take supernatant spare, centrifuge tube blots weighs that its is heavy after residual liquid again, the front and back pipe method of double differences twice Value is thallus weight in wet base.Weight in wet base calculation formula are as follows:

Thallus weight in wet base (g/L)=(upper pigging weight-centrifugation front tube weight is removed after centrifugation)/bacterium solution volume

OD₆₀₀The measurement of value: the wavelength regulation of ultraviolet specrophotometer is subjected to light absorption value measurement at 600nm, when with this Section supernatant of bacteria solution is as control, and replication 3 times using average value as OD₆₀₀Measurement result.

(2) BCA measures total protein concentration

1. before the assay, by BCA reagent A and B, 50:1 prepares appropriate BCA working solution by volume, and mixes well.

2. with protein standard substance (5mg/ml) is completely dissolved, and it is spare to be diluted to final concentration of 0.5mg/ml.

3. standard items are added in 96 orifice plates by 0,1,2,4,6,8,12,16,20 μ l, and every hole standard items are mended with PBS Enough to 20 μ l.

4. taking 20 μ l to be added in 96 plate holes in the sample after dilution

5. 200 μ lBCA working solutions are added in every hole, after 37 DEG C of placement 30min, light absorption value is measured at 562nm.

6. drawing standard curve according to standard items light absorption value, sample total protein concentration is calculated

The purifying of the fusion protein CYGB-PL of the present invention of embodiment 7

The extraction of sample total protein

Recombinant is fermented, fermentation liquid is extracted, 6000rpm is centrifuged 25min, removes thallus and residue, takes supernatant It is spare.

Ammonium sulfate precipitation

Total protein supernatant after taking 6 equal portions to be centrifuged, every part of 10m1 are slowly added to the solid sulphuric acid of different quality into liquid Ammonium, respectively to 10%, 20%, 30%, 40%, 50%, 60%, 70% and 80% saturation degree, be slowly stirred uniformly, 4 DEG C of ice Case stands 6h and saltouts.12000rpm is centrifuged 30min at 4 DEG C, collects supernatant and precipitating, the precipitating 5mM of 10mL respectively Tris-HCl (pH 8.8) buffer solution, the fibrinolytic of respective supernatant and precipitating is detected using fibrin plate method, Solusphere diameter is measured, and draws grade ammonium sulfate salting-out curve.

The best ammonium sulfate saturation degree of salt fractionation purifying fibrinolysin is determined according to the fibrinolytic of supernatant and precipitating.Hair Solid ammonium sulfate (preferably being ground before addition, keep stirring in adition process) is slowly added in zymotic fluid to 30% saturation Degree, stirs evenly, and 4 DEG C of refrigerators stand 6h, 12000rpm, 4 DEG C of centrifugation 30min, and removal precipitating is collected supernatant, continuously added Solid sulphuric acid is pressed to 80% saturation degree, and 4 DEG C of refrigerators stand 6h, 12000rpm, 4 DEG C of centrifugation 30min, collect precipitating.Precipitating is used slow Fliud flushing 5mM Tris-HCl (pH 8.8) sufficiently dissolves, and carries out next step desalination.

G-25 Ago-Gel desalination

Chromatographic column is loaded using Sephadex G-25, first with the equilibration buffer 5mM Tris-HCl of 3 times of bed volumes (pH 8.8) sufficiently balanced gel column, the protein sample dissolution equilibrium buffer 5mM Tris-HCl (pH after then saltouing 8.8) in, chromatographic column is passed through with 2mL/min after 0.22 μm of membrane filtration, then elute egg with 5mM Tris-HCl (pH 8.8) White, flow velocity 2mL/min collects first peak.

Ion-exchange chromatography

Chromatographic column is loaded using DEAE Sepharose Fast Flow, first with the equilibration buffer of 5 times of bed volumes 5mM Tris-HCl (pH 8.8) balance, the Peak Activity for then collecting upper step desalination is with 2mL/min loading, with identical after loading Buffer 5mM Tris-HCl (pH 8.8) elutes unadsorbed protein.Again with 5mM Tris-HCl (pH 8.8)~ 125mM NaCl -5mM Tris-HCl (pH 8.8) carries out gradient elution, and the every pipe of flow velocity 2mL/min, 5min is collected.Ultraviolet inspection The ultraviolet absorption value at instrument detection 280nm wavelength is surveyed, and by the situation of change of recorder record light absorption value.Measure each collecting pipe Protein concentration and enzyme activity, and according to enzyme activity collect component.

Influence of 8 pH of embodiment to enzyme stability

Using the extensive buffer of 2.2~pH of pH 10.9, preparation method is as follows:

50mM citric acid 200mL is prepared, when with NaOH tune pH to 3.0,4.0,5.0 and 6.0, takes out 2mL buffer respectively It is fitted into test tube.

Prepare 50mM Tris 200mL, with HCl adjust pH be 7.0,8.0 and 9.0 when, respectively take out 2mL buffer be packed into In test tube.

50mM glycine 200mL is prepared, when with NaOH tune pH to 10.0 and 11.0,2mL buffer is taken out respectively and is packed into examination Guan Zhong.

With the enzyme solutions of the final concentration of 1mg/mL of the buffer of different pH value, while 37 DEG C of placements are placed in, it will after 1h PH of buffer is adjusted to 7.4, and with the variation of fibrin plate method measurement enzyme activity, 37 DEG C of constant incubators place 18h, with highest Enzyme activity is opposite enzyme activity 100%, and every group of data set three Duplicate Samples and are averaged.

Influence of 9 temperature of embodiment to enzyme stability

PL solution is prepared with PBS buffer solution (10mM, pH 7.4), concentration 1mg/mL is respectively placed in 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 1h, 2h, 4h and 8h are kept the temperature at 80 DEG C, separately sampled, 37 DEG C of constant incubators place 18h, in fibre The variation of fibrillarin plate measurement enzyme activity.It is opposite enzyme activity 100% with highest enzyme activity, every group of data set three Duplicate Samples and make even Mean value

The influence of 10 metal ion of embodiment and enzyme inhibitor to enzyme activity

Different metal ions M g will be contained²⁺(MgSO₄·7H₂O)、Cu²⁺(CuCl₂)、Ca²⁺(CaCl₂)、K⁺(KCl)、Na⁺ (NaCl) reagent and enzyme inhibitor PMSF and EDTA prepare the solution for becoming 2mM with 10mM PBS buffer solution.By PL and solution Mixing, makes the final concentration of 1mg/mL of PL, and after 37 DEG C of heat preservation 30min, the variation of enzyme activity is measured with fibrin plate method, 37 DEG C of constant incubators of plate place 18h, not add the enzyme solution vigor of metal ion or enzyme inhibitor for 100%, every group of number It is averaged according to three Duplicate Samples are set.

Activation of the embodiment 11 to plasminogen

The standard fibers albumen plate of preparation contains plasminogen, referred to as positive plate.By dressing plate in Place 30min in 85 DEG C of baking ovens, after condensation be free of the plate of activities of endothelial tissue plasminogen, referred to as negative plate^[77].It is added Whether the 100 μ L of urokinase of 800U/mL has remaining plasminogen as positive control, to verify in plate.Purified egg White (concentration 1mg/mL) 100 μ L is put on negative plate and positive plate, and it is big that hydrolytic circle is observed after 37 DEG C of heat preservation 18h It is small.

The mode of action of the embodiment 12 to fibrinogen

After taking 400 μ L enzyme solutions (1mg/mL) and 100 μ L bovine fibrinogen solution (6mg/mL) to be sufficiently mixed, it is placed in 37 DEG C of incubation reactions, it is fast respectively at 0,1min, 3min, 5min, 10min, 30min, 60min, 120min, 180min and 240min Speed sampling, ice bath terminate reaction.Be added isometric 2 mercapto ethanol to sample, boiling water bath 5min, to the albumen after denaturation into Glue 5% is concentrated in row SDS-PAGE electrophoretic analysis, resolving gel concentration 12%, observes the degradation of fibrinogen A α, B β, γ chain Situation.

The external thrombolytic experiment of embodiment 13

(1) blood 5mL is taken to be placed in serum tube from the eyeball of mouse, 4 DEG C of placement natural coagulations blot clot table with filter paper Face weighs 100mg native mouse whole blood sludged blood respectively, is added in three test tubes.

(2) experiment sets negative control physiological saline group, positive control urokinase group (1000U/mL), experimental group CYGB-PL (1000U/mL) and experimental group PL (1000U/mL), sample potency press the calibration of urokinase unit of activity standard curve.

(3) it is separately added into the corresponding solution of 2mL in the test tube that clot has been added, is placed in 37 DEG C of constant-temperature tables and shakes slowly (60r/min).Test tube upper solution smear, microscopically observation red cell morphology and quantity are drawn every 1h and are photographed to record. Slowly the quality of remaining blood clot in each test tube is weighed after shaking 3h, and calculates dissolution of blood clot rate.Dissolution of blood clot rate=(before dissolution Blood clot quality after blood clot quality-dissolution)/dissolve preceding blood clot quality.

Thrombolytic experiment in 14 body of embodiment

Mouse tail thrombus model is caused using carrageenan.Select clinically used urokinase injection as positive right According to negative control is physiological saline group, and experimental group is PL enzyme solution and CYGB-PL enzyme solution.Each tested group of enzyme activity unit is unified to be used Urokinase standard items Fibrinolytic Activity standard curve is demarcated.

Daily 20,000~40,000 unit of urokinase injection clinical treatment acute cerebral thrombosis and peripheral artery vein embolism, once gives Medicine can be calculated the medication of 25g mouse according to 9 times that the dosage of people and animal dose conversion table (60kg/ people) mouse is people Dosage range is 75~150U.Therefore, it is 4000U/kg that experiment positive control, which is selected as injection urokinase dosage,；Low dosage administration Group, injection dosage 4000U/kg；High dose administration group, injection dosage 8000U/kg；Thrombus model group and Normal group, The amount of injecting normal saline is 8mL/kg, and the above solution is prepared with 0.9%NaCl, and sample potency presses urokinase unit of activity Standard curve calibration.

(1) male mice in kunming 32, age of mouse are 5 weeks, 30 ± 2g of weight, are mentioned by University of Fuzhou's Wu Shi animal center For.Mouse feeding environment is 23 ± 2 DEG C of raising temperature, humidity 50~60%, illumination 12h.It is randomly divided into 7 groups, i.e. normal control Group (5), high dose PL administration group (5), low dosage PL administration group (5), high dose CYGB-PL administration group (5), low dose Amount CYGB-PL administration group (5), urokinase positive controls (5) and thrombus model group (5), adaptable fed 1 week；

(2) 1% carrageenan 160mg/kg is injected intraperitoneally afterwards for 24 hours, observes tail portion thrombosis situation afterwards for 24 hours.

(3) carrageenin injection for 24 hours after, low dosage administration group, injection dosage 4000U/kg；High dose administration group, note Penetrating dosage is 8000U/kg；Urokinase positive controls, the dosage for injecting urokinase is 4000U/kg；Thrombus model group and normal Group, the amount of injecting normal saline are 8mL/kg.Intraperitoneal injection is primary, continuous injection three days.Experimental result GraphPad Prism software is counted, and is usedIt indicates.

The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e., Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.

Sequence table

<110>permitted Ruian

<120>fusion protein of a kind of cytoglobin and Sipunculus nudus plasmin

<160> 10

<210> 1

<211> 453

<212>

<213>

<220>

<221> PRO

<222>

<223>

<400> 1

Met Glu Lys Val Pro Gly Glu Met Glu Ile Glu Arg Arg Glu Arg

5 10 15

Ser Glu Glu Leu Ser Glu Ala Glu Arg Lys Ala Val Gln Ala Met

20 25 30

Trp Ala Arg Leu Tyr Ala Asn Cys Glu Asp Val Gly Val Ala Ile

35 40 45

Leu Val Arg Phe Phe Val Asn Phe Pro Ser Ala Lys Gln Tyr Phe

50 55 60

Ser Gln Phe Lys His Met Glu Asp Pro Leu Glu Met Glu Arg Ser

65 70 75

Pro Gln Leu Arg Lys His Arg Cys Arg Val Met Gly Ala Leu Asn

80 85 90

Thr Val Val Glu Asn Leu His Asp Pro Asp Lys Val Ser Ser Val

95 100 105

Leu Ala Leu Val Gly Lys Ala His Ala Leu lys His Lys Val Glu

110 115 120

Pro Val Tyr Phe Lys Ile Leu Ser Gly Val Ile Leu Glu Val Val

125 130 135

Ala Glu Glu Phe Ala Ser Asp Phe Pro Pro Glu Thr Gln Arg Ala

140 145 150

Trp Ala lys Leu Arg Gly Leu Ile Tyr Ser His Val Thr Ala Ala

155 160 165

Tyr Lys Glu Val Gly Trp Val Gln Gln Val Pro Asn Ala Thr Thr

170 175 180

Pro Pro Ala Thr Leu Pro Ser Ser Gly Pro Arg Ser Lys lys Arg

185 190 195

Gly Gly Gly Gly Ser lys Arg Arg Lys Ile Glu Gly Arg Arg Pro

200 205 210

Pro Gly Phe Ile Val Gly Gly Arg Pro Ala Gly Lys Gly Arg Trp

215 220 225

Pro Trp Gln Leu Ser Leu Gln Ile Glu Gly Ile Gly Trp Gly Trp

230 235 240

His Thr Cys Gly Ala Ile Leu Leu Gly Ala Asn Arg Ala Leu Thr

245 250 255

Ala Ala His Cys Thr Glu Gly Arg Ser Gly Phe Arg Ile Leu Ala

260 265 270

Gly Ala Ser Asn Ile Gly Ala Ser Pro Asp His Glu Ala Glu Ser

275 280 285

Leu Val Ser Ser Thr Thr Glu His Pro Gly Phe Asp Arg Phe Ala

290 295 300

Pro Gly Ile Pro Asn Asp Val Gly Thr Leu Ala Leu Ala Thr Ala

305 310 315

Val Asn Ala Gly Gly Ala Ile Ala Tyr Ala Ser Leu Ala Pro Thr

320 325 330

Gly Gly Pro Asn Tyr Ala Gly Asn Glu Cys Trp Ala Ser Gly Trp

335 340 345

Gly Arg Leu His Gly Asp Asn Gly Pro Leu Pro Asp Gln Leu Gln

350 355 360

Glu Val Arg Ile Asp Ala Leu Thr Asn Ala Glu Cys Arg Ser Arg

365 370 375

Met Pro Ile Asn Leu Gln Glu Asn Val Leu Asp Gln His Ile Cys

380 385 390

Ile His Gly Asn Gly Asn Gln Gly Ala Cys Gln Gly Asp Ser Gly

395 400 405

Gly Pro Leu Asn Cys Arg Asp Gly Ser Phe Met Val Val Gly Val

410 415 420

Thr Ser Trp Val Val Gly Ser Met Asp Ser Ser Cys Met Thr Glu

425 430 435

Tyr Pro Asn Val Tyr Ala Arg Val Ser His Phe Arg Ser Trp Ile

440 445 450

Asp Ser Asn

451 452 453

<210> 2

<211> 240

<212>

<213>

<220>

<221> PRO

<222>

<223>

<400> 2

Ile Val Gly Gly Arg Pro Ala Gly Lys Gly Arg Trp Pro Trp Gln

5 10 15

Leu Ser Leu Gln Ile Glu Gly Ile Gly Trp Gly Trp His Thr Cys

20 25 30

Gly Ala Ile Leu Leu Gly Ala Asn Arg Ala Leu Thr Ala Ala His

35 40 45

Cys Thr Glu Gly Arg Ser Gly Phe Arg Ile Leu Ala Gly Ala Ser

50 55 60

Asn Ile Gly Ala Ser Pro Asp His Glu Ala Glu Ser Leu Val Ser

65 70 75

Ser Thr Thr Glu His Pro Gly Phe Asp Arg Phe Ala Pro Gly Ile

80 85 90

Pro Asn Asp Val Gly Thr Leu Ala Leu Ala Thr Ala Val Asn Ala

95 100 105

Gly Gly Ala Ile Ala Tyr Ala Ser Leu Ala Pro Thr Gly Gly Pro

110 115 120

Asn Tyr Ala Gly Asn Glu Cys Trp Ala Ser Gly Trp Gly Arg Leu

125 130 135

His Gly Asp Asn Gly Pro Leu Pro Asp Gln Leu Gln Glu Val Arg

140 145 150

Ile Asp Ala Leu Thr Asn Ala Glu Cys Arg Ser Arg Met Pro Ile

155 160 165

Asn Leu Gln Glu Asn Val Leu Asp Gln His Ile Cys Ile His Gly

170 175 180

Asn Gly Asn Gln Gly Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu

185 190 195

Asn Cys Arg Asp Gly Ser Phe Met Val Val Gly Val Thr Ser Trp

200 205 210

Val Val Gly Ser Met Asp Ser Ser Cys Met Thr Glu Tyr Pro Asn

215 220 225

Val Tyr Ala Arg Val Ser His Phe Arg Ser Trp Ile Asp Ser Asn

230 235 240

<210> 3

<211> 192

<212>

<213>

<220>

<221> PRO

<222>

<223>

<400> 3

Met Glu Lys Val Pro Gly Glu Met Glu Ile Glu Arg Arg Glu Arg

5 10 15

Ser Glu Glu Leu Ser Glu Ala Glu Arg Lys Ala Val Gln Ala Met

20 25 30

Trp Ala Arg Leu Tyr Ala Asn Cys Glu Asp Val Gly Val Ala Ile

35 40 45

Leu Val Arg Phe Phe Val Asn Phe Pro Ser Ala Lys Gln Tyr Phe

50 55 60

Ser Gln Phe Lys His Met Glu Asp Pro Leu Glu Met Glu Arg Ser

65 70 75

Pro Gln Leu Arg Lys His Arg Cys Arg Val Met Gly Ala Leu Asn

80 85 90

Thr Val Val Glu Asn Leu His Asp Pro Asp Lys Val Ser Ser Val

95 100 105

Leu Ala Leu Val Gly Lys Ala His Ala Leu lys His Lys Val Glu

110 115 120

Pro Val Tyr Phe Lys Ile Leu Ser Gly Val Ile Leu Glu Val Val

125 130 135

Ala Glu Glu Phe Ala Ser Asp Phe Pro Pro Glu Thr Gln Arg Ala

140 145 150

Trp Ala lys Leu Arg Gly Leu Ile Tyr Ser His Val Thr Ala Ala

155 160 165

Tyr Lys Glu Val Gly Trp Val Gln Gln Val Pro Asn Ala Thr Thr

170 175 180

Pro Pro Ala Thr Leu Pro Ser Ser Gly Pro Arg Ser

185 190

<210> 4

<211> 16

<212>

<213>

<220>

<221> PRO

<222>

<223>

<400> 4

Lys lys Arg Gly Gly Gly Gly Ser lys Arg Arg Lys Ile Glu Gly Arg

5 10 15

<210> 5

<211> 5

<212>

<213>

<220>

<221> PRO

<222>

<223>

<400> 5

Arg Pro Pro Gly Phe

1 2 3 4 5

<210> 6

<211> 1376

<212>

<213>

<220>

<221> DNA

<222>

<223>

<400> 6

ctcgagaaga gaatggagaa agtgccaggc gagatggaga tcgagcgcag ggagcggagc 60

gaggagctgt ccgaggcgga gaggaaggcg gtgcaggcta tgtgggcccg gctctatgcc 120

aactgcgagg acgtgggggt ggccatcctg gtgaggttct ttgtgaactt cccctcggcc 180

aagcagtact tcagccagtt caagcacatg gaggatcccc tggagatgga gcggagcccc 240

cagctgcgga agcacgcctg ccgagtcatg ggggccctca acactgtcgt ggagaacctg 300

catgaccccg acaaggtgtc ctctgtgctc gcccttgtgg ggaaagccca cgccctcaag 360

cacaaggtgg aaccggtgta cttcaagatc ctctctgggg tcattctgga ggtggtcgcc 420

gaggaatttg ccagtgactt cccacctgag acgcagagag cctgggccaa gctgcgtggc 480

ctcatctaca gccacgtgac cgctgcctac aaggaagtgg gctgggtgca gcaggtcccc 540

aacgccacca ccccaccggc cacactgccc tcttcggggc cgggatccaa gaagagaggt 600

ggtggaggtt ccaagagaaa gagaattgaa ggtagacgtc cacctggttt catcgtgggt 660

ggacgtccag caggaaaggt cgttggccat ggcagctgtc cctgcagatc gaaggaattg 720

gttggggtca tacctgcgga gctatcctgc tgggagcaaa cagagctctg accgcagctc 780

attgcaccga aggtcgttcc ggtttcagaa tcctggcagg agcttccaac attggtgcat 840

cccctgatca tgaagctgaa tccctggtgt cctccaccac cgaacatcca ggatttgatc 900

gtttcgcacc aggtatccca aacgatgttg gtactctggc tctggcaacc gctgttaacg 960

caggaggtgc aatcgcttac gcatccctgg cacctaccgg aggtccagat tacgctggta 1020

acgaatgctg ggcttccgga tggggtagac tgcatggaga taacggtcca ctgccagatc 1080

agctgcagga agtgcgtatt gatgcactga ccaacgctga atgcagatcc cgtatgccta 1140

tcaacctgca ggaaaacgtg ctggatcagc atatctgcat tcatggaaac ggtaaccagg 1200

gtgcatgcca gggagattcc ggaggtccac tgaactgcag agatggttcc ttcatggttg 1260

tgggagttac ctcctgggtg gttggttcca tggattcctc ctgcatgacc gaatacccaa 1320

acgtgtacgc tcgtgtgtcc cattttagat cctggatcga ttccaactaa gaattc 1376

<210> 7

<211> 726

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 7

atcgtgggtg gacgtccagc aggaaaaggt cgttggccat ggcagctgtc cctgcagatc 60

gaaggaattg gttggggtca tacctgcgga gctatcctgc tgggagcaaa cagagctctg 120

accgcagctc attgcaccga aggtcgttcc ggtttcagaa tcctggcagg agcttccaac 180

attggtgcat cccctgatca tgaagctgaa tccctggtgt cctccaccac cgaacatcca 240

ggatttgatc gtttcgcacc aggtatccca aacgatgttg gtactctggc tctggcaacc 300

gctgttaacg caggaggtgc aatcgcttac gcatccctgg cacctaccgg aggtccagat 360

tacgctggta acgaatgctg ggcttccgga tggggtagac tgcatggaga taacggtcca 420

ctgccagatc agctgcagga agtgcgtatt gatgcactga ccaacgctga atgcagatcc 480

cgtatgccta tcaacctgca ggaaaacgtg ctggatcagc atatctgcat tcatggaaac 540

ggtaaccagg gtgcatgcca gggagattcc ggaggtccac tgaactgcag agatggttcc 600

ttcatggttg tgggagttac ctcctgggtg gttggttcca tggattcctc ctgcatgacc 660

gaatacccaa acgtgtacgc tcgtgtgtcc cattttagat cctggatcga ttccaactaa 720

gaattc 726

<210> 8

<211> 642

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 8

ctcgagaaga gaatggagaa agtgccaggc gagatggaga tcgagcgcag ggagcggagc 60

gaggagctgt ccgaggcgga gaggaaggcg gtgcaggcta tgtgggcccg gctctatgcc 120

aactgcgagg acgtgggggt ggccatcctg gtgaggttct ttgtgaactt cccctcggcc 240

aagcagtact tcagccagtt caagcacatg gaggatcccc tggagatgga gcggagcccc 300

cagctgcgga agcacgcctg ccgagtcatg ggggccctca acactgtcgt ggagaacctg 360

catgaccccg acaaggtgtc ctctgtgctc gcccttgtgg ggaaagccca cgccctcaag 420

cacaaggtgg aaccggtgta cttcaagatc ctctctgggg tcattctgga ggtggtcgcc 480

gaggaatttg ccagtgactt cccacctgag acgcagagag cctgggccaa gctgcgtggc 540

ctcatctaca gccacgtgac cgctgcctac aaggaagtgg gctgggtgca gcaggtcccc 600

aacgccacca ccccaccggc cacactgccc tcttcggggc cg 642

<210> 9

<211> 54

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 9

ggatccaaga agagaggtgg tggaggttcc aagagaaaga gaattgaagg taga 54

<210> 10

<211> 15

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 10

cgtccacctg gtttc 15

Claims

1. the fusion protein of a kind of cytoglobin and Sipunculus nudus plasmin, entitled CYGB-PL, the fusion protein have Amino acid sequence shown in SEQ ID NO:1.

2. fusion protein as described in claim 1, which is characterized in that Sipunculus nudus plasmin has shown in SEQ ID NO:2 Amino acid sequence.

3. fusion protein as described in claim 1, which is characterized in that cytoglobin has ammonia shown in SEQ ID NO:3 Base acid sequence.

4. fusion protein as described in claim 1, which is characterized in that the fusion protein contains flexible polypeptide, flexible polypeptide tool There is amino acid sequence shown in SEQ ID NO:4.

5. fusion protein as described in claim 1, which is characterized in that the fusion protein contains fibrin ferment pentapeptide, fibrin ferment five Peptide has amino acid sequence shown in SEQ ID NO:5.

6. a kind of engineering bacteria, entitled pPIC-9-CYGB-PL-GS115 express fusion protein described in claim 1 CYGB-PL。

7. a kind of recombinant vector, entitled pPIC-9-CYGB-PL.

8. recombinant vector as claimed in claim 7, which is characterized in that its preparation process includes: through BglII single endonuclease digestion, and electricity is transferred to Yeast GS115.

9. the pharmaceutical composition containing fusion protein described in claim 1.

10. fusion protein described in claim 1 is preparing the application in antithrombotic reagent.